Yuejun Wang1,2, Zhi Jia1, Shu Diao2, Xiao Lin2, Xiaomeng Lian3, Liping Wang2, Rui Dong4, Dayong Liu5,6, Zhipeng Fan7. 1. Department of Endodontics, Tianjin Medical University School of Stomatology, Tianjin, China. 2. Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, China. 3. Department of Stomatology, Beijing Shijitan hospital, Capital Medical University, Beijing, China. 4. Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, China. dongrui95@126.com. 5. Department of Endodontics, Tianjin Medical University School of Stomatology, Tianjin, China. dyliuperio@tmu.edu.cn. 6. Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, China. dyliuperio@tmu.edu.cn. 7. Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, China. zpfan@ccmu.edu.cn.
Abstract
OBJECTIVES: Mesenchymal stem cell (MSC)-mediated tissue regeneration represents a promising strategy for repair of tissue defects, but its molecular mechanisms remain unclear, restricting the use of MSCs. Our previous study indicated that insulin-like growth factor-binding protein 5 (IGFBP5) exerted a valuable effect on osteogenic differentiation of MSCs, but its molecular mechanisms underlying directed differentiation remained unclear. In this study, we have investigated the molecular role of IGFBP5 in regulating this osteogenic differentiation potential. MATERIALS AND METHODS: Periodontal ligament stem cells (PDLSCs) were isolated from periodontal ligament tissue. Wharton's jelly of umbilical cord stem cells (WJCMSCs) was obtained commercially. Lentiviral IGFBP5 shRNA was used to silence IGFBP5. Retroviruses expressing wild-type IGFBP5 were used to overexpress IGFBP5 in the WJCMSCs. Recombinant human IGFBP5 protein (rhIGFBP5) was used to treat PDLSCs for 24 h. Western blot analysis was used to detect the MAPK signalling pathway, and alkaline phosphatase (ALP) activity, Alizarin Red staining and quantitative calcium analysis were used to study osteogenic differentiation potentials. RESULTS: Overexpression of IGFBP5 or rhIGFBP5 increased expression levels of phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated mitogen-activated protein kinase 1 and 2 (p-MEK1/2) and phosphorylated extracellular regulated protein kinases (p-Erk1/2) in both WJCMSCs and PDLSCs. Consistently, silenced IGFBP5 was found to effectively inhibit expression of p-JNK, p-Erk1/2 and p-MEK1/2 in PDLSCs and WJCMSCs. Furthermore, inhibition of JNK by its inhibitor, SP600125, or MEK/Erk signalling by its inhibitor, PD98059, dramatically blocked IGFBP5-enhanced ALP activity and in vitro mineralization in both PDLSCs and WJCMSCs. CONCLUSIONS: Our results demonstrated that IGFBP5 promoted osteogenic differentiation potentials of PDLSCs and WJCMSCs via the JNK and MEK/Erk signalling pathways.
OBJECTIVES: Mesenchymal stem cell (MSC)-mediated tissue regeneration represents a promising strategy for repair of tissue defects, but its molecular mechanisms remain unclear, restricting the use of MSCs. Our previous study indicated that insulin-like growth factor-binding protein 5 (IGFBP5) exerted a valuable effect on osteogenic differentiation of MSCs, but its molecular mechanisms underlying directed differentiation remained unclear. In this study, we have investigated the molecular role of IGFBP5 in regulating this osteogenic differentiation potential. MATERIALS AND METHODS: Periodontal ligament stem cells (PDLSCs) were isolated from periodontal ligament tissue. Wharton's jelly of umbilical cord stem cells (WJCMSCs) was obtained commercially. Lentiviral IGFBP5 shRNA was used to silence IGFBP5. Retroviruses expressing wild-type IGFBP5 were used to overexpress IGFBP5 in the WJCMSCs. Recombinant humanIGFBP5 protein (rhIGFBP5) was used to treat PDLSCs for 24 h. Western blot analysis was used to detect the MAPK signalling pathway, and alkaline phosphatase (ALP) activity, Alizarin Red staining and quantitative calcium analysis were used to study osteogenic differentiation potentials. RESULTS: Overexpression of IGFBP5 or rhIGFBP5 increased expression levels of phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated mitogen-activated protein kinase 1 and 2 (p-MEK1/2) and phosphorylated extracellular regulated protein kinases (p-Erk1/2) in both WJCMSCs and PDLSCs. Consistently, silenced IGFBP5 was found to effectively inhibit expression of p-JNK, p-Erk1/2 and p-MEK1/2 in PDLSCs and WJCMSCs. Furthermore, inhibition of JNK by its inhibitor, SP600125, or MEK/Erk signalling by its inhibitor, PD98059, dramatically blocked IGFBP5-enhanced ALP activity and in vitro mineralization in both PDLSCs and WJCMSCs. CONCLUSIONS: Our results demonstrated that IGFBP5 promoted osteogenic differentiation potentials of PDLSCs and WJCMSCs via the JNK and MEK/Erk signalling pathways.
Authors: Robert S Flynn; Sunila Mahavadi; Karnam S Murthy; John M Kellum; John F Kuemmerle Journal: Am J Physiol Gastrointest Liver Physiol Date: 2009-10-01 Impact factor: 4.052